Fuxin
Zhang
,
Tao
Yang
,
Kailing
Yang
,
Ruixi
Zhou
,
Yu
Zhang
,
Wenwen
Chen
,
Zhetong
Liu
,
Guanqun
Zhan
* and
Zengjun
Guo
*
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an 710061, P. R. China. E-mail: zhanguanqun@mail.xjtu.edu.cn; guozj@mail.xjtu.edu.cn
First published on 28th November 2022
Twelve new Cephalotaxus alkaloids (1–12) and nine known analogues (13–21) were isolated and identified from the twigs and leaves of Cephalotaxus sinensis. The structures of the new compounds (1–12) were elucidated by extensive spectroscopic analysis and single-crystal X-ray diffraction analysis. Cephalosine H (8) is the third example of an alkaloid containing the cephalolancine skeleton. Cephalosines J and K (10 and 11) are the rare natural Δ(2)1-alkene-6-hydroxyl homoerythrina-type alkaloids isolated from the Cephalotaxus genus. The racemization of cephalotaxine-type alkaloids is discussed. Alkaloids 6, 7, 11, 16, 18 and 19 exhibited broad and potent cytotoxicities against five human cancer cell lines, with IC50 values ranging from 0.053 to 10.720 μM, highlighting these compounds as promising leads for the development of new antitumor agents.
Cephalotaxus sinensis (Rehder & E.H.Wilson) H.L.Li, a major species found in China and rich in resources, is used as a traditional folk medicine in China to treat inflammation, dyspepsia, ascariasis, and cough.8,9 Previous phytochemistry investigations of C. sinensis, concerning its characteristic Cephalotaxus alkaloids, are limited,10,11 reminding us that there may be more new alkaloids with significant antitumor activity waiting to be discovered.
In the present study, the twigs and leaves of C. sinensis collected from Qinling Mountains have been phytochemically investigated, leading to the isolation of twenty-one compounds (Fig. 1), including seven new cephalotaxine-type alkaloids (1–7) containing two pairs of enantiomers (1 and 2), one new cephalolancine-type alkaloid (8), four new homoerythrina-type alkaloids (9–12), and nine known analogues (13–21). Herein, the isolation, structure determination, and cytotoxicity and SARS-CoV-2 3CLpro inhibitory activities of compounds 1–21 are described.
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Fig. 3 (a) ECD spectra of 1a and 1b; (b) ECD spectra of 2a and 2b; (c) ECD spectra of 6, 7, and 17–19. |
No. | 1 | 2 | 3 | 4 | 5 |
---|---|---|---|---|---|
1 | 6.68, s | 5.01, s | 4.70, br s | 1.91, dd (14.0, 9.0) | 2.26, dd (15.4, 6.2) |
1.78, dd (14.0, 9.0) | 1.53, br d (15.4) | ||||
2 | 3.86, t (9.0) | 3.54, d (6.2) | |||
3 | 4.70, d (9.2) | 3.99, dd (5.3, 0.5) | |||
4 | 3.67, s | 3.64, d (9.2) | 3.2 d (5.3) | 3.12 s | 3.34, overlap |
6 | 2.05, ddd (12.5, 12.5, 8.8) | 1.98, ddd (12.2, 10.8, 9.0) | 1.79, 2H, overlap | 1.73, m | 1.73, 2H, m |
1.99, ddd (12.5, 8.8, 3.2) | 1.86, ddd (12.2, 9.0, 3.0) | 1.62, m | |||
7 | 1.94, m | 1.79, m | 1.79, overlap | 1.64, 2H, m | 1.68, 2H, m |
1.79, m | 1.70, m | 1.71, m | |||
8 | 2.95, ddd (9.5, 9.5, 3.4) | 2.90, ddd (9.5, 9.5, 3.5) | 2.53, ddd (9.2, 8.5, 2.8) | 2.65, ddd (9.0, 8.5, 3.4) | 2.67, dd (9.0, 8.5, 3.5) |
2.75, ddd (9.5, 9.5, 7.5) | 2.62, overlap | 2.44, q (9.2) | 2.32, dq (9.0, 2.2) | 2.33, dq (9.0, 2.2) | |
10 | 2.85, ddd (11.8, 11.8, 7.8) | 2.84, ddd (11.5, 11.5, 7.8) | 2.78, 2H, overlap | 2.80, dd (12.5, 4.0) | 2.77, dd (12.6, 4.0) |
2.55, dd (11.8, 8.0) | 2.62, overlap | 2.73, d (12.5) | 2.64, d (12.6) | ||
11 | 2.47, dd (15.0, 7.8) | 3.36, ddd (14.5, 11.5, 7.8) | 4.76, dd (4.0, 2.0) | 4.92, br d (4.0) | 4.86, overlap |
2.37, ddd (15.0, 11.8, 8.0) | 2.28, dd (14.5, 7.0) | ||||
14 | 6.75, s | 6.73, s | 6.71, s | 6.75, s | 6.75, s |
17 | 6.67, s | 6.61, s | 6.80, s | 6.78, s | 6.76, s |
18 | 5.91, 2H, brs | 5.94, d (1.1) | 5.93, d (1.0) | ||
5.92, d (1.1) | 5.92, d (1.0) | ||||
19 | 4.02, 2H, dq (7.0, 2.8) | ||||
20 | 1.39, 3H, t (7.0) | ||||
2-OCH3 | 3.70, 3H, s | 3.71, 3H, s | 3.50, 3H, s | 3.42, 3H, s | |
15-OCH3 | 3.83, 3H, s | ||||
16-OCH3 | 3.85, 3H, s |
No. | 1 | 2 | 3 | 4 | 5 | 8 | 9 | 10 | 11 | 12 |
---|---|---|---|---|---|---|---|---|---|---|
1 | 126.8 | 98.0 | 97.6 | 31.7 | 32.4 | 39.4 | 130.2 | 135.1 | 135.1 | 117.8 |
2 | 158.7 | 163.3 | 167.3 | 85.9 | 86.4 | 79.2 | 132.2 | 130.8 | 130.7 | 33.4 |
3 | 203.8 | 74.7 | 78.3 | 104.8 | 106.9 | 66.5 | 74.4 | 76.5 | 76.6 | 76.0 |
4 | 60.9 | 58.8 | 50.0 | 55.8 | 54.6 | 130.9 | 27.8 | 37.4 | 37.2 | 39.2 |
5 | 67.0 | 72.5 | 75.5 | 66.4 | 67.9 | 147.8 | 66.1 | 71.5 | 71.2 | 70.5 |
6 | 39.5 | 44.0 | 34.5 | 39.1 | 39.6 | 69.5 | 70.0 | 80.2 | 80.2 | 144.3 |
7 | 20.8 | 20.9 | 22.4 | 20.9 | 20.9 | 39.3 | 61.2 | 38.4 | 38.4 | 28.6 |
8 | 53.8 | 54.5 | 50.3 | 50.6 | 50.9 | 25.5 | 55.8 | 48.2 | 48.2 | 47.5 |
9 | 50.1 | |||||||||
10 | 48.8 | 49.8 | 53.5 | 55.2 | 55.5 | 50.4 | 43.3 | 43.5 | 51.2 | |
11 | 32.0 | 32.0 | 78.9 | 77.0 | 77.0 | 46.5 | 30.0 | 29.1 | 29.1 | 24.5 |
12 | 132.0 | 134.7 | 130.5 | 132.1 | 131.9 | 32.5 | 36.8 | 34.1 | 33.8 | 37.8 |
13 | 130.4 | 128.6 | 128.2 | 127.5 | 128.3 | 130.1 | 136.5 | 135.3 | 134.9 | 136.3 |
14 | 113.7 | 118.1 | 116.9 | 110.8 | 110.8 | 132.0 | 135.5 | 139.4 | 138.5 | 132.1 |
15 | 148.7 | 147.1 | 148.6 | 148.4 | 148.2 | 115.2 | 112.1 | 112.4 | 117.2 | 117.1 |
16 | 147.8 | 146.6 | 148.5 | 148.7 | 148.7 | 147.6 | 146.4 | 146.8 | 147.6 | 146.3 |
17 | 111.2 | 118.2 | 109.9 | 106.9 | 106.9 | 147.8 | 147.2 | 147.2 | 148.6 | 146.9 |
18 | 102.4 | 102.3 | 102.3 | 117.1 | 112.4 | 111.6 | 115.9 | 120.2 | ||
19 | 67.1 | 102.2 | 102.2 | |||||||
20 | 14.7 | |||||||||
2-OCH3 | 57.6 | 57.8 | 58.8 | 58.1 | 57.3 | |||||
3-OCH3 | 57.0 | 56.6 | 56.7 | 56.3 | ||||||
15-OCH3 | 56.9 | |||||||||
16-OCH3 | 56.8 | 56.5 | 56.8 | 56.8 | ||||||
17-OCH3 | 56.5 |
Cephalosine B (2) has a molecular formula of C18H23NO4, as determined using the [M + H]+ ion at m/z 318.1704 (calcd for C18H24NO4, 318.1705) in the HRESIMS and 13C NMR data. Analysis of its NMR data (Tables 1 and 2) revealed that compound 2 corresponded well to 14,13 except for the presence of resonances for an oxymethyl group (δH 3.83, 3H, s; δC 56.9) in 2 and the absence of resonances for a methylenedioxy moiety in 14 (δH 5.86, 1H, d, J = 1.2 Hz; δH 5.85, 1H, d, J = 1.2 Hz; δC 102.1). The methoxy group was located at C-15 as supported by the HMBC correlation from 15-OCH3 (δH 3.83) to C-15 (δC 147.1) along with the NOESY correlation from 15-OCH3 (δH 3.83) to H-14 (δH 6.73). The relative configurations of 2 were established by analysis of the proton–proton coupling constants and the NOESY spectrum. The correlations of H-1 (δH 5.01) with H-6β (δH 1.86) and of H-4 (δH 3.64) with H-6α (δH 1.98)/H-3 (δH 4.70)/H-14 (δH 6.73) combined with the same coupling constant between H-3 and H-4 (J3,4 = 9.2 Hz) with those of co-isolated cephalotaxine-type alkaloids (16–19) suggested that the cephalotaxine core in 2 has the same relative configurations as those of 16–19.3,6 However, the optical rotation data of 2 were much smaller than those of the analogues (16–19) suggesting that 2 may be a pair of enantiomers (2a and 2b), and was purified and confirmed in the same way as 1 (Fig. 2b). Considering the similar ECD spectra of 2a and cephalofortine C, and the opposite ECD spectra of 2b and cephalofortine C,14 the absolute configurations of 2a and 2b were defined to be 3S,4S,5R and 3R,4R,5S, respectively.
Compound 3, a colorless crystal, has a molecular formula of C18H21NO4 as established using its HRESIMS (m/z 316.1551 [M + H]+, calcd 316.1549) and 13C NMR data. The NMR data of 3 (Tables 1 and 2) closely resembled those of 15,15 except for a methoxy group (δH 3.85, 3H, s; δC 56.8) in 3 instead of a methylenedioxy moiety (δH 5.92, 1H, s; δH 5.91, 1H, s; δC 102.3) in 15. These were verified from the HMBC correlation between the methoxy group (δH 3.85) and C-16 (148.5) along with the NOESY correlations observed from the methoxy group (δH 3.85) to H-17 (δH 6.80). Finally, the single crystals of 3 were obtained from a MeOH/H2O (20:
1) solution. The absolute configuration of each stereogenic center in 3 was defined to be 3S,4S,5R,11R by Ga Kα single-crystal X-ray crystallographic data analysis with a Flack parameter of 0.11 (5) (Fig. 4).
The molecular formula of cephalosine D (4) was determined to be C18H21NO5 on the basis of HRESIMS (m/z 332.1504 [M + H]+, calcd 332.1498) and 13C NMR data. The 1D NMR data (Tables 1 and 2) of 4 closely resembled those of cephalotine B,16 with the main difference being the presence of a methoxy group (δH 3.50, 3H, s; δC 58.8) in 4 replacing the C-2 hydroxyl group in cephalotine B, which was confirmed by the HMBC correlation from the methoxy group to C-2 (δC 85.9). Thus, 4 was an etherified derivative of cephalotine B. Finally, the absolute configuration of 4 was defined as 2R,3S,4S,5S,11R based on single-crystal X-ray diffraction analysis (Fig. 4).
Compound 5 has the same molecular formula of C18H21NO5 as that of 4, determined using the same [M + H]+ ion at m/z 332.1486 (calcd for C18H22NO5, 332.1498) in the HRESIMS and 13C NMR data. The NMR data of 5 showed high similarities to those of 4 (Tables 1 and 2), with the only difference being the signals vicinal to C-2, indicating the β-orientation of H-2. The β-orientation of H-2 was determined based on the coupling constant between H-1 and H-2 in 5 (J1,2 = 6.2 Hz),16 differing from that of 4 (J1,2 = 9.0 Hz) with an α-orientation of H-2. The NOESY cross-peaks from 2-OCH3 (δH 3.42) to H-1α (δH 1.53) and H-6α (δH 1.73) further supported the elucidation. Thus, the structure of cephalosine E (5) was determined as shown.
Compounds 6 and 7 share the same molecular formula, C29H31NO9, as determined using the negative-ion peak at m/z [M − H]− 536.1932 and 536.1933, respectively (calcd for C29H30NO9, 536.1926). The 1H and 13C NMR data of compounds 6 and 7 (Table 3) were similar to those of the co-isolated compounds 18 and 19, respectively, with the differences being restricted to the substituents on the C-2′ position. The signals in 1H and 13C NMR spectra established the presence of a phenmethyl moiety in 6 (δH 7.21, m, 2H, H-3′′/H-7′′; 7.20, m, 2H, H-4′′/H-6′′; 7.19, m, 1H, H-5′′; 2.82, d, J = 14.1 Hz, 1H, H-1a′′; 2.73, d, J = 14.1 Hz, 1H, H-1b′′; δC 137.0, C-2′′; 131.7, C-3′′/C-7′′; 128.9, C-4′′/C-6′′; 127.7, C-5′′; 40.9, C-1′′) and 7 (δH 7.22, m, 2H, H-3′′/H-7′′; 7.21, m, 2H, H-4′′/H-6′′; 7.18, m, 1H, H-5′′; 3.07, d, J = 13.8 Hz, 1H, H-1a′′; 2.80, d, J = 13.8 Hz, 1H, H-1b′′; δC 136.9, C-2′′; 132.0, C-3′′/C-7′′, 128.9, C-4′′/C-6′′; 127.8, C-5′′; 42.4, C-1′′), which was further confirmed by the HMBC correlations from H-1′′ to C-2′′/C-3′′/C-7′′. The connectivity of the phenmethyl moiety at C-2′ in 6 and 7 was established on the basis of HMBC cross-peaks from H-1′′ to C-1′/C-2′/C-3′. Therefore, the relative configurations of 6 and 7 were deduced to be the same as those of 18 and 19, respectively. The absolute configurations of 6 and 7 were determined based on similar ECD curves as those of 18 and 19, respectively (Fig. 3c). The structures of 18 and 19 were almost identical except for the configuration at C-3′, while the ECD curve of 18 differed from 19 with only a minor difference at around 225 nm, a negative Cotton effect in 18 and a positive Cotton effect in 19, indicating that the Cotton effect at around 225 nm was a characteristic of the configuration at C-3′ in cephalotaxine esters. The ECD spectrum of 18, whose absolute configuration was determined to be 3S,4S,5R,2′R,3′R by Ga Kα single-crystal X-ray diffraction analysis (Fig. 4), was in good agreement with a previous report,3 further supporting the deduction. Therefore, the absolute configurations of 6 and 7 were determined to be the same as those of 18 (3S,4S,5R,2′R,3′R) and 19 (3S,4S,5R,2′R,3′S), respectively, based on their similar ECD data (Fig. 3c).
No. | 6 | 7 | ||
---|---|---|---|---|
δ H | δ C | δ H | δ C | |
1 | 5.23, s | 101.2 | 5.23, s | 101.2 |
2 | 159.9 | 160.0 | ||
3 | 5.81, d (9.7) | 76.7 | 5.92, d (9.8) | 76.5 |
4 | 3.88, d (9.7) | 56.9 | 3.88, d (9.8) | 56.5 |
5 | 72.3 | 72.1 | ||
6 | 2.00, ddd (12.5, 10.5, 9.0) | 44.1 | 2.02, ddd (12.5, 10.5, 9.0) | 44.0 |
1.94, ddd (12.5, 9.0, 3.0) | 1.94, ddd (12.5, 9.0, 3.0) | |||
7 | 1.83, m | 21.0 | 1.83, m | 20.9 |
1.74, m | 1.74, m | |||
8 | 2.96, ddd (9.5, 9.5, 3.8) | 54.8 | 2.96, ddd (9.5, 9.5, 3.8) | 54.7 |
2.65, dd (9.5, 7.0) | 2.65, dd (9.5, 7.0) | |||
10 | 2.91, ddd (11.8, 11.8, 7.0) | 49.9 | 2.87, ddd (11.8, 11.8, 7.0) | 49.7 |
2.69, dd (11.8, 8.0) | 2.63, dd (11.8, 8.0) | |||
11 | 3.36, ddd (14.2, 11.8, 8.0) | 32.6 | 3.15, ddd (14.1, 11.8, 8.0) | 32.2 |
2.55, dd (14.2, 7.0) | 2.39, dd (14.1, 7.0) | |||
12 | 134.7 | 134.9 | ||
13 | 129.8 | 130.1 | ||
14 | 6.63, s | 114.4 | 6.60, s | 114.6 |
15 | 148.5 | 148.4 | ||
16 | 147.7 | 147.3 | ||
17 | 6.74, s | 111.2 | 6.69, s | 111.1 |
18 | 5.89, d (1.2) | 102.2 | 5.90, d (1.2) | 102.3 |
5.77, d (1.2) | 5.79, d (1.2) | |||
1′ | 173.1 | 173.5 | ||
2′ | 81.6 | 81.1 | ||
3′ | 3.17, s | 76.3 | 3.26, s | 75.7 |
4′ | 173.0 | 173.1 | ||
1′′ | 2.82, d (14.1) | 40.9 | 3.07, d (13.8) | 42.4 |
2.73, d (14.1) | 2.80, d (13.8) | |||
2′′ | 137.0 | 136.9 | ||
3′′ | 7.21, m | 131.7 | 7.22, m | 132.0 |
4′′ | 7.20, m | 128.9 | 7.21, m | 128.9 |
5′′ | 7.19, m | 127.7 | 7.18, m | 127.8 |
6′′ | 7.20, m | 128.9 | 7.21, m | 128.9 |
7′′ | 7.21, m | 131.7 | 7.22, m | 132.0 |
2-OCH3 | 3.63, 3H, s | 57.9 | 3.72, 3H, s | 57.8 |
The determination of the absolute configuration at C-3′ in cephalotaxine-type alkaloids has been an attractive and challenging work for a long time. Two reliable methods, chemical methods and CD experiments, were developed to solve the problem. The chemical methods including Mosher's method and chemical transformations were applied for the determination of the absolute configuration, such as cephalezomines C and D,17 cephastigiamide A,18 and cephaloverines E and F;3 however this method is limited by the amount of the compounds. The CD spectrum of the molybdate complex of the diacid derived from the hydrolysis of isoharringtonine was first used to determine the absolute configuration of its side chain;19 then this method was applied for 3′S-hydroxyneoharringtonine,20 while the presence of other vicinal diols or carboxyl groups restrict the application of this method. In this study, we observed that the Cotton effect at around 225 nm in the ECD spectra is a characteristic for determining the absolute configuration at C-3′ in cephalotaxine esters (excludes nitrogen oxides). It should be a simple and convenient method, especially for compounds with low molecular weight.
Cephalosine H (8) was obtained as a white amorphous powder with a molecular formula of C19H25NO4 from its HRESIMS (m/z 332.1865 [M + H]+, calcd for C19H26NO4,332.1862) and 13C NMR data. The NMR data of 8 (Tables 2 and 4) were similar to those of cephalounei A,21 except for the presence of an additional methoxy group (δH 3.81, 3H, s; δC 56.5) in 8 and the absence of a methylenedioxy moiety (δH 5.89, 1H, s; 5.88, 1H, s; δC 102.4) in cephalounei A. The additional methoxy group was located at C-16 based on the HMBC correlation from the methoxy group (δH 3.81) to C-16 (δC 147.6) and the NOESY correlation from the methoxy group (δH 3.81) to H-15 (δH 6.65). Furthermore, 2D NMR experiments including HSQC, HMBC, 1H–1H COSY, and NOESY established the structure of 8 (Fig. 5). The absolute configuration of 8 was elucidated to be the same as that of cephalounei A based on their similar ECD and optical rotation data.21 Thus, the absolute configuration of 8 was assigned to be 2R,3S,6S, and named cephalosine H.
No. | 8 | 9 | 10 | 11 | 12 |
---|---|---|---|---|---|
1 | 1.99, dd (13.5, 8.5) | 5.89, br d (10.0) | 5.70, dd (10.2, 1.7) | 5.72, dd (10.3, 2.0) | 5.58, br s |
1.77, overlap | |||||
2 | 3.65, ddd (8.5, 3.5, 2.5) | 6.07, dd (10.0, 4.5) | 5.90, ddd (10.2, 1.7, 1.7) | 5.94, ddd (10.3, 2.0, 2.0) | 2.64, m |
1.99, m | |||||
3 | 4.36, t (3.8) | 3.92, t (4.5) | 3.79, m | 3.81, ddd (9.0, 5.0, 2.0) | 3.29, m |
4 | 5.69, d (3.8) | 3.25, d (14.5) | 2.15, dd (13.2, 5.2) | 2.19, dd (13.3, 5.0) | 2.78, dd (11.5, 3.2) |
1.75, dd (14.5, 4.5) | 2.08, dd (13.2, 9.0) | 2.11, dd (13.3, 9.0) | 1.49, t (11.5) | ||
7 | 1.77, overlap | 3.61, br s | 1.94, ddd (12.0, 9.0, 9.0) | 1.94, ddd (12.1, 9.0, 9.0) | 2.46, m |
1.68, m | 1.70, ddd (12.0, 6.5, 1.2) | 1.71, ddd (12.1, 6.3, 1.2) | 2.31, m | ||
8 | 1.75, m | 3.22, d (12.0) | 3.06, ddd (9.0, 9.0, 1.2) | 3.09, ddd (9.0, 9.0, 1.2) | 2.74, ddd (9.0, 8.9, 7.0) |
1.62, m | 2.75, d (12.0) | 2.89, overlap | 2.91, overlap | 2.70, overlap | |
9 | 2.85, ddd (9.2, 6.7, 2.7) | ||||
2.80, ddd (9.2, 9.0, 6.4) | |||||
10 | 3.12, ddd (13.0, 12.5, 2.0) | 3.00, dd (15.0, 8.3) | 3.02, dd (15.0, 8.0) | 3.45, ddd (14.6, 12.9, 2.0) | |
2.81, ddd (13.0, 3.5, 3.5) | 2.89, overlap | 2.91, overlap | 3.14, ddd (14.6, 4.4, 3.2) | ||
11 | 3.41, ddd (15.0, 12.5, 4.0) | 1.83, m | 2.12, m | 2.16, m | 1.83, m |
2.96, ddd (15.0, 5.4, 3.0) | 1.69, m | 1.79, m | 1.82, m | 1.57, m | |
12 | 3.16, ddd (17.7, 12.5, 5.4) | 3.35, t (15.0) | 2.93, ddd (14.5, 12.2, 4.0) | 2.98, ddd (14.5, 12.3, 4.3) | 3.11, ddd (15.2, 12.5, 1.1) |
2.76, ddd (17.7, 4.0, 3.0) | 2.70, dd (15.0, 4.5) | 2.51, ddd (14.5, 4.0, 4.0) | 2.55, ddd (14.5, 4.3, 4.3) | 2.70, overlap | |
15 | 6.65, s | 6.76, s | 6.87, s | 6.97, s | 6.73, s |
18 | 6.52, s | 6.59, s | 6.54, s | 6.65, s | 6.62, s |
19 | 5.85, 2H, s | 5.85, 2H, s | |||
2-OCH3 | 3.46, 3H, s | ||||
3-OCH3 | 3.05, 3H, s | 3.30, 3H, s | 3.32, 3H, s | 3.21, 3H, s | |
16-OCH3 | 3.81, 3H, s | 3.78, 3H, s | 3.73, 3H, s | ||
17-OCH3 | 3.80, 3H, s |
Cephalosine I (9) has a molecular formula of C19H21NO4 as deduced from its positive ion peak at m/z 328.1551 [M + H]+ (calcd for 328.1543, C19H22NO4). The 1D NMR data (Tables 2 and 4) of 9 showed high similarity to those of the co-isolated compound fortunine (20),22 except for the signals vicinal to C-3, indicating 9 was a C-3 epimer of 20. The absolute configuration of 9 was defined as 3S,5R,6S,7R by Ga Kα X-ray crystallographic data analysis (Fig. 4).
Cephalosine J (10), existing as colorless crystals, possesses the molecular formula of C19H23NO4 as determined using the HRESIMS (m/z 330.1710 [M + H]+, calcd for C19H24NO4, 330.1705) and 13C NMR data. The NMR data of 10 resembled those of 3-epi-schellhammericine,23 except for the significant downfield shifts of C-6 (δC 80.2), C-7 (δC 38.4), C-1 (δC 135.1), and C-5 (δC 71.5) in 10, suggesting that a hydroxyl group was attached to C-6 in 10. To determine the configuration of the hydroxyl group at the quaternary carbon C-6, the crystals of 10 were obtained from a MeOH/H2O (20:
1) solution. Therefore, the absolute configuration of 10 was determined to be 3R,5R,6S by the Ga Kα X-ray crystallographic data analysis with a Flack parameter of 0.06 (3) (Fig. 4).
Cephalosine K (11) possesses a molecular formula of C20H27NO4 as established using the HRESIMS peak at m/z 346.2021 [M + H]+ (calcd for 346.2013, C20H28NO4). The 1H and 13C NMR data of 11 (Tables 2 and 4) resembled those of 10 except for the absence of a methylenedioxy moiety in 10 and the presence of two additional methoxy groups (δH 3.78, 3H, s; δC 56.8 and δH 3.80, 3H, s; δC 56.5) in 11. The HMBC correlation from the methoxy group (δH 3.78) to C-16 (δC 147.6) together with the key NOESY correlation of the methoxy group (δH 3.78) and H-15 (δH 6.97) in 11 indicate that the later methoxy group was attached at C-16. The other methoxy group (δH 3.80, s; δC 56.5) attached to C-17 was determined by the same procedure. Hence, the absolute configuration of 11 was determined to be the same as that of 10 and verified using the similar ECD spectra of these two compounds.
Though the planar structures of these two compounds has been reported as reduction products of comosimine and 3-epi-wilsonine, respectively, the absolute configurations of the hydroxyl at C-6 in 10 and 11 has not been determined.24–26 In this work, the absolute configuration of the hydroxyl group at C-6 in homoerythrina-type alkaloids 10 and 11 was determined by single X-ray crystallographic data analysis.
Cephalosine L (12) has the molecular formula C19H25NO3 based on the HRESIMS ion at m/z 316.1917 [M + H]+ (calcd for 316.1913, C19H26NO3) and 13C NMR. The NMR data of compound 12 closely resembled those of 21,27 except for slight differences in the chemical shifts of the aromatic ring, suggesting a methoxy to be attached to C-16 in 12, instead of C-17 in 21. The HMBC correlation from 16-OCH3 (δH 3.73) to C-16 (δC 146.3), along with the key NOESY correlation from 16-OCH3 (δH 3.73) to H-15 (δH 6.73) in 12 indicated the connection of the methoxy group with C-16. Therefore, the structure of 12 was defined as shown in Fig. 1 and named cephalosine L.
The known alkaloids 13–21 were identified as cephalotaxinone (13),12 cephalotaxine (14),13 3-deoxy-3,11-epoxy-cephalotaxine (15),15 neoharringtonine (16),20 isoharringtonine (17),28 cephaloverine E (18),3 5′-des-O-methylisoharringtonine (19),29 fortunine (20),30 and taxodine (21),27 respectively, via comparison of the reported spectroscopic data.
As shown in Fig. 6 and Fig. S1,† (−) cephalotaxine (14a) and (−) cephalosine B (2a) were not observed to be converted into their enantiomers after being heated at 75 °C in 95% ethanol for 9 hours, suggesting that the racemization was not generated in the process of extraction. These two compounds were then dispersed in hydrochloric acid solution (pH = 2) for 24 h, followed by placing them in an alkaline solution (pH = 9) for 24 h. Interestingly, racemization was not observed under these reaction conditions. Moreover, racemization did not occur during silica gel column chromatography and HPLC analysis. Considering these results, we suggest that the racemization of cephalotaxine-type alkaloids is not generated in the process of extraction and isolation but it exists in nature.
Compounds | IC50![]() |
||||
---|---|---|---|---|---|
HL-60 | A549 | SMMC-7721 | MDA-MB-231 | SW480 | |
a Compound 17 was not detected, other compounds, IC50 > 40 μM. b Values were expressed as means ± SD (n = 3). c Positive control. | |||||
1a | >40 | 33.750 ± 0.630 | 17.050 ± 0.160 | 26.340 ± 0.830 | 29.960 ± 1.340 |
6 | 0.400 ± 0.005 | 0.840 ± 0.011 | 1.066 ± 0.086 | 1.773 ± 0.043 | 3.904 ± 0.233 |
7 | 2.236 ± 0.583 | 0.727 ± 0.020 | 0.945 ± 0.014 | 0.878 ± 0.057 | 1.452 ± 0.037 |
11 | 8.428 ± 1.210 | 5.556 ± 0.318 | 6.272 ± 0.198 | 10.320 ± 0.540 | 10.720 ± 0.930 |
16 | 0.133 ± 0.005 | 0.053 ± 0.007 | 0.054 ± 0.001 | 0.151 ± 0.008 | 0.133 ± 0.005 |
18 | 0.053 ± 0.011 | 1.663 ± 0.303 | 3.030 ± 0.073 | 3.347 ± 0.239 | 4.942 ± 0.184 |
19 | 0.604 ± 0.087 | 0.188 ± 0.163 | 0.180 ± 0.009 | 0.494 ± 0.042 | 0.438 ± 0.050 |
HHTc | 0.027 ± 0.002 | 0.029 ± 0.002 | 0.120 ± 0.006 | 0.077 ± 0.005 | 0.066 ± 0.003 |
Cisplatinc | 4.156 ± 0.189 | 9.104 ± 0.300 | 4.732 ± 0.543 | 14.030 ± 0.750 | 26.340 ± 0.830 |
Paclitaxelc | <0.008 | <0.008 | 0.123 ± 0.008 | <0.008 | <0.008 |
Footnote |
† Electronic supplementary information (ESI) available: NMR, HRESIMS, UV, and ECD spectra. CCDC 2213948–2213954. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d2ob01980a |
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